The present invention relates to a novel hyaluronic acid gel with fluidity or with fluidity and transparency and a method of its production, and further, to a biomedical material with good biocompatibility.
Hyaluronic acid (hereinafter referred to simply as HA) is a linear macromolecular polysaccharide consisting of alternately bonded xcex2-D-N-acetylglucosamine and xcex2-D-glucuronic acid. HA is found not only in connective tissues of mammals but also in cockscombs and the capsules of Streptococci. HA is obtainable not only by extraction from cockscombs and umbilical cords, but also as purified products from the culture broth of streptococci.
Natural HA is polydisperse in respect of molecular weight and is known to show excellent biocompatibility even when implanted or injected into the body by virtue of the absence of species and organ specificity. However, because of the relatively short in vivo residence time of HA solution in biological application, improvement of the persistency of HA by chemical crosslinking with various chemical modifiers has been attempted to broaden its use for medical materials.
(I) Concerning the joints, synovial fluid supplies nutrition to the articular cartilage and has incomparable functions as a lubricant and a shock absorber. It is clarified that its excellent viscoelastisity heavily owes to one of the main components, HA.
Concentration and molecular weight analyses of HA demonstrated the concentration and molecular weight of HA in the synovial fluid from patients with arthritis such as osteoarthritis and chronic articular rheumatism generally tended to lower than in normal synovial fluid, and the lower concentration and molecular weight of HA were closely associated with development of locomotor dysfunction and pain attributable to the weaker lubricating action and the weaker protecting action on the surface of the articular cartilage of synovial fluid.
Injection of high molecular weight HA solution (Artz: from Seikagaku Corporation, average molecular weight 900000; Hyalgan: from Fidia, average molecular weight  less than 500000) into diseased joints has been widely adopted as an effective measure for osteoarthritis among those articular diseases, and the source of high purity HA preparations for this purpose is cockscombs. Such HA preparations from cockscombs are biologically inherent and quite safe but usually have to be administered as frequently as several to 10 times to show significant therapeutic effect.
Persistency tests on rabbits revealed that HA with a molecular weight of less than 1000000 administered into the knee joint cavities disappeared from the knee joint cavities in 1 to 3 days and suggested the need of frequent administrations (Blood Coagulation and Fibrinolysis, vol 12, 173, 1992).
On the other hand, the molecular weight of HA found in the living body is reported to be as high as millions to 10000000, and a crosslinked HA derivative [Hylan: from Biomatrix] obtained by treatment with a chemical crosslinker has been developed as a therapeutic agent for knee joints with the idea that high molecular weight HA closer to the biologically intact one is likely to have higher effect.
Reportedly, the crosslinked HA persisted for a period as long as 20 to 30 days after administration into rabbit knee joint cavities in the above-mentioned persistency tests and produced sufficient effect when administered three times in clinical tests, and is practically used as a therapeutic agent for arthritis (Journal of Theumatology vol.20, 16, 1993).
(II) Next, concerning emboli, treatments through embolization are known to effective for various diseases such as angiopathy, paraplastic aneurysm and varix. Obstruction of arteries as the nourishing channels for tumours is also effective in tumour treatment.
Some proposals have been made for embolization. For example, a balloon embolization method using a balloon-tip catheter has been developed (W. Taki et al., Surg. Neurol, Vol.12, 363, 1979). In addition, a method in which 2-hydroxyethyl methacrylate (HEMA) is introduced into a balloon together with a polymerization catalyst through a catheter is also known (W. Taki et al., Surg. Neurol, Vol.13, 140, 1980).
For cancer treatment through embolization, use of cisplatin-containing chitin (Tahara et al., Cancer and Chemotherapy, vol.21(13), 2225, 1994), use of poly(benzyl 1-glutamate) microspheres carrying cisplatin (Li C et al., Parm, Res., Vol.11(12), 1792, 1994) and use of SMANCS and Lipiodol suspension together with gelatin sponge as a embolizing material (Nakamura et al., Cancer and Chemotherapy, vol.22(11), 1390, 1996) have been reported. In addition, Poly(DL-lactate) microspheres are reported as a suitable material for use in embolismic chemotherapy in combination with continuous injection of a chemotherapeutic agent (Flandroy P et al., J Control Release, Vol.44(2/3), 153, 1997) while it is mentioned that they have to biodegrade in a couple of days so that when this therapy is practiced repeatedly.
There are a lot of problems such as the short time obstruction in the balloon embolization due to shriveling of the balloon as a bar to production of satisfactory effect and the possibility of polymerization of monomers such as HEMA inside the catheter. Most embolizing materials used in embolismic chemotherapy are synthetically available and hardly biodegradable and doubtful in respect of biocompatibility. Poly(DL-lactate) microspheres, though biodegradable, do not guarantee complete safety when repeatedly administered.
Though highly biocompatible HA has no problem with safety, HA does not embolize when merely administered in the form of solution, and is required to have improved local persistency.
(III) Concerning soft tissues, the idea of injecting various materials to repair or swell soft tissues has rapidly developed since the invention of the subcutaneous injection needle, and a number of materials have been injected into human bodies to remedy soft tissues and skins. Among them, liquid silicone has been used widely for injection but is not used as much recently as it used to be due to its side effects such as skin ulceration attributable to its long retention time. Collagen has also been injected so far in various forms such as chemically crosslinked forms and fibrous forms. Crosslinked solid collagen requires incision to be injected and has problems plasticity and flexibility. There is a disclosure about fibrous collagen in U.S. Pat. No. 3949073.
However, it shrinks in volume as its liquid components are absorbed and has to be supplemented. Injectable types of collagen like this can hardly be freed of contaminants such as immunity substances, are costly and do not necessarily have appropriate physical properties.
HA has also been attempted as an injection for soft tissues (Ann. Plast. Surg., Vol.38, 308, 1997). Because HA in solution is rapidly absorbed in vivo, various methods for chemical crosslinking of HA have been attempted to improve persistency and retention in soft tissues (U.S. Pat. No. 4582865, JP-B-6-37575, JP-A-7-97401, JP-A-60-130601).
And hylan B gel is commercially available as Hylaform in Europe (The Chemistry Biology and Medical Application of Hyaluronan and its Derivatives Vol.72, p278, PORTLAND PRESS).
(IV) Next, reference will be made to the posterior part of the eyeball, especially the retina bordered on the vitreous body. The retina marks the posterior boundary of the intraocular space, while the lens and the ciliary body mark the anterior boundary. The retina consists of two layers, the receptor layer of photosensitive cells in contact with the vitreous humor and the layer of pigment epithelial cells adjacent to the choroid. Liquid infusion into the receptor layer causes retinal detachment, separating the two layers of the retina.
For treatment of retinal detachment, the peeled retina is brought into contact with the pigmented epithelial layer and fastened by photocoagulation or cryocoagulation. The contact is achieved by pressing a inward buckle against the sclera and the choroids from outside or by generating pressure from vitreous humor onto the retina through volume expansion of vitreous humor by injection.
In the latter case, because vitreous humor has to be removed partly or completely due to too much spilt blood for reabsorption or inward growth of the retina accompanying retinal detachment, various materials have been attempted as artificial vitreous bodies.
These artificial vitreous bodies are intended to maintain the shape of the eyeball and bring back the retina in position while pressing the retina against the pigmented epithelium in the vitreous chamber.
As artificial vitreous bodies, physiological saline, glycerin, animal vitreous bodies, air, various gases, polyvinyl alcohol, collagen gel, silicone oil, HA and perfluorocarbons may be mentioned, and air, gases such as sulfur hexafluoride, silicone oil, liquid perfluorocarbons such as perfluorooctane and perfluorodecalin are generally used now.
Various expansive gases are used as artificial vitreous bodies by themselves or in mixtures with air, and have proven to be useful (American Journal of ophthalmology, Vol.98, 180, 1984).
However, they sometimes cause complications such as increase of intraocular pressure and coreclisis attributable to gas expansion or keratoleukoma attributable to their contact with the corneal endothelium and impose on patients a heavy burden of keeping their faces down for a long time.
Silicone oil maintains the intraocular space for a longer time than gases by virtue of its little absorbability and accelerates adhesion of the retina effectively (Retina, Vol.7, 180, 1987), but is used with the proviso that it is drawn out after exertion of the pressing effect on the retina. Further, it is said to have serious problems of cataract, glaucoma and toxic effects on the ocular tissue (Ophthalmology, Vol.27, 1081, 1985).
Liquid perfluorocarbons as artificial vitreous bodies are proved to cause complications such as proliferative vitreoretinopathy, cataract and intraocular hypotension and are reported to be more questionable than silicone oil and gases in respect of safety and effectiveness (New Ophthalmology, Vol.12, 1053, 1995).
HA has been investigated a lot since Balazs reported its application in the field of ophthalmology (Mod. Probl. Ophthalmol., Vol.10, 3 1972) and is widely used in ophthalmic surgery, especially intraocular implantation.
HA is inherently biogenic and never induces toxic or immunological reactions. However, HA can not exert the effect of maintaining the intraocular space for a long time sufficient for treatment of serious retinal detachment because HA injected into the vitreous chamber dissolves in aqueous humor and is discharged from the eye through the anterior chamber and the fibrous trabecular goniomeshwork without being decomposed.
Though vitreous injections containing HA, for example, which contain at least 1.5 wt %, preferably from 2 to 2.5 wt % of HA with a molecular weight of at least 900000, preferably 1600000 to 2000000 are disclosed in JP-A-5-184663, they are not retained in the intraocular space {Nippon Ganka Kiyou, vol.38, 927, 1987}. Additionally, over 1 wt % solution of HA with such a molecular weight strains a syringe when ejected from the syringe into the vitreous body and is not practical.
As mentioned above, improvement of the in vivo retention of HA is essential for its applications, and various chemical crosslinkers have been used to crosslink HA (U.S. Pat. No. 4582865, JP-A-60-13060, JP-A-63-28166, JP-B-6-37575, JP-B-6-69481, JP-A-7-97401, JP-A-7-59303). Further, production of a photo-crosslinked HA gel by irradiation of a photo-crosslinkable HA derivative is also known (JP-A-143604).
However, these cross-linked products of HA are not what is called HA any longer, and among the desired properties for materials used in vivo, non-toxicity and non-immunogenicity can not absolutely be secured for them considering procedures for removal of crosslinkers and the difficulty of complete denial of the presence of residual crosslinkers.
The present inventors have found out a simple method of producing a hardly water soluble HA gel made of hyaluronic acid alone for the first time (PCT/JP98/03536). However, the gel is sheet-like, filmy, flaky, spongy or massive and lacks fluidity.
Therefore, the present inventors have proposed a HA gel slurry obtained by suspending granules or flakes of the hardly water soluble HA gel in aqueous solution with an idea that a hardly water soluble HA-containing material with fluidity would be useful and find various medical applications. The HA gel slurry has fluidity and is easy to eject from an injector including it.
To take the advantages of the excellent biocompatibility which HA inherently has by itself to the maximum, hardly water soluble HA gels with fluidity obtainable without using any chemical crosslinker or modifier are favorable. But such gels have not been developed yet, and only a HA gel slurry obtained by suspending the hardly water soluble HA gel flakes in aqueous solution has been proposed.
Besides, there is a problem that the necessity to crush the hardly water soluble HA gel by using ultrasonic waves or a mixer in preparation of the HA gel adds up the production steps.
In general, transparency is also desired in view of quality control. On the other hand, for use of HA gels in the field of ophthalmology, especially as artificial vitreous bodies, fluidity is required in view of handling properties, while transparency is required in view of effectiveness. Further, gels with refractive indices closer to that of the vitreous body are preferable (1.3345-1.3348; (Ganka Shinryo Practice, Vol.22, pp234, 1996, Bunkodo, Tokyo). However, no gels have been developed yet that have these properties.
The present inventors thought that a hardly water soluble and transparent HA gel would be obtained by imparting fluidity of HA solution to a hardly water soluble HA gel made of HA alone obtained without using any crosslinker or the like and broaden the applications of HA gels and as a result of extensive research for such a gel, have accomplished the present invention.
Namely, the present invention provides (1) a gel made of HA alone which is hardly soluble in a neutral aqueous solution and has fluidity enough to be easily ejectable from an injector, (2) the HA gel according to (1), which dissolves in a neutral aqueous solution at 37xc2x0 C. in 12 hours to a degree of dissolution of at most 50%, (3) the HA gel according to (1), which dissolves to yield solubilized HA having a branched structure and partly containing a molecular weight fraction with a branching degree of at least 0.5, when treated under accelerating conditions for acid hydrolysis of HA, (4) the HA gel according to any one of (1) to (3), which is transparent, (5) a method of producing the HA gel according to (4), which comprises adjusting a HA aqueous solution containing an inorganic salt to pH 3.5 or below, and freezing and thawing the solution, (6) a biomedical material containing a gel made of HA alone which satisfies the following requirements (a) and (b): (a) the HA gel dissolves in a neutral aqueous solution at 37xc2x0 C. in 12 hours to a degree of dissolution of at most 50%, and (b) the gel dissolves to yield solubilized HA having a branched structure and partly containing a molecular weight fraction with a branching degree of at least 0.5, when treated under accelerating conditions for acid hydrolysis of HA, (7) a biomedical material containing a HA gel and un-gelled HA, wherein the HA gel dissolves in a neutral aqueous solution at 37xc2x0 C. in 12 hours to a degree of at most 50%, and the HA gel dissolves to yield solubilized HA having a branched structure and partly containing a molecular weight fraction with a branching degree of at least 0.5, when treated under accelerating conditions for acid hydrolysis of HA, (8) the biomedical material according to (6) or (7), which is an injection for treatment of arthrosis, (9) the biomedical material according to (6) or (7), which is an embolizing material, (10) the biomedical material according to (6) or (7), which is an injection for a soft tissue, and (11) the biomedical material according to (6) or (7), which is an artificial vitreous body.
Now, the present invention will be described below in detail.
In the present invention, HA obtained by extraction from animal tissues or by fermentation may be used without any restriction on its source.
The strain used in fermentation is preferably a HA-producing microorganism isolated from nature such as the genus Streptococcus or a mutant which steadily produces HA in high yield such as Streptococcus equi FM-100 (accession number 9027 given by National Institute of Bioscience and Human-Technology) disclosed in JP-A-63-123392 or Streptococcus equi FM-300 (accession number 2319 given by National Institute of Bioscience and Human-Technology) disclosed in JP-A-2-234689. Pure HA obtained from cultures of the above-mentioned mutants may be used.
The molecular weight of the HA to be used in the present invention is preferably within the range of from about 1xc3x97105 to about 1xc3x97107 Da. HA having a higher molecular weight may also be used after the molecular weight is lowered into this range by treatment such as hydrolysis.
In the present invention, the concept of HA is used so as to include its alkali metal salts such as sodium, potassium and lithium salts, too.
In the present invention, by HA alone, it is meant that no chemical crosslinker or chemical modifier is used other than HA, that HA is not in the form of a complex with a cationic polymer, and that the gel is an auto-crosslinked gel.
The HA gel according to the present invention is a polymer having a three dimensional network structure or its swollen product. The three dimensional network structure is made of crosslinked HA.
In the present invention, the difficulty in dissolution is defined by the solubility in a neutral aqueous solution at 37xc2x0 C. and means that the gel dissolves in a neutral aqueous solution at 37xc2x0 C. in 12 hours to a degree of dissolution of at most 50%, preferably at most 30%, particularly preferably at most 10%.
The HA gel according to the present invention can be solubilized through degradation by treatment under accelerating conditions for acid hydrolysis of HA. When the solubilized HA retains the crosslinked structure, it is distinguished as branched HA from linear HA according to the theory of polymer solution.
The accelerating conditions for acid hydrolysis of HA according to the present invention are preferably such that the pH of the aqueous solution is 1.5 and the temperature is 60xc2x0 C. It is well known that cleavage of the main chain of HA through hydrolysis of glycosidic bonds is remarkably accelerated in an acidic or alkaline aqueous solution as compared with that in a neutral aqueous solution. In addition, acid hydrolysis is accelerated at a higher temperature.
In the present invention, the molecular weights and branching degrees of the fractions separated by GPC according to molecular weight are measured on-line continuously by the GPC-MALLS method. In the present invention, the branching degree was measured by the elution volume method which compares the molecular weight of each fraction of the solubilized HA with the molecular weight of a fraction at the same elution volume of linear HA as a control. The branching degree is the number of branch points in one polymer chain of the solubilized HA and plotted against the molecular weight of the solubilized HA. Measurement of the branching degree by the GPC-MALLS method by the elution volume method is described in PCT/JP98/03536.
Solubilized HA was diluted with the GPC eluent for concentration adjustment and filtered through a membrane filter of 0.2 xcexcm before measurement.
If the HA gel according to the present invention has a crosslinked structure which is stable under accelerating conditions for acid hydrolysis of HA, a branched structure is recognized in the solubilized HA according to the theory of polymer solution. The HA gel according to the present invention has a branching degree of at least 0.5.
In the present invention, by easily ejectable from an injector, it is meant that the HA gel of the present invention can be ejected at room temperature about 25xc2x0 C. at a rate of 0.1 ml/sec with a force of at most 50 N when loaded into a disposable syringe (hereinafter referred to as an injector) of 2.5 to 3 ml with an inner diameter of about 1 cm equipped with a disposable injection needle of 21 G with an outer diameter of about 0.8 mm.
In the present invention, transparency means that the visible light transmittance of the HA gel of the present invention in a spectrometric cuvette of 10 mm thick measured at 340 nm to 800 nm is at least 50%, preferably 70%, particularly preferably 90%, based on the transmittance of water.
For pH adjustment of a HA aqueous solution, any acid that can adjust the pH to 3.5 or below may be used. Preferably, a strong acid such as hydrochloric acid, nitric acid and sulfuric acid is used to decrease the amount of an acid.
As the inorganic salt to be added to a HA solution in the present invention, a salt of a monovalent metal such as a sodium salt or a potassium salt, or a salt of a bivalent metal such as a magnesium salt, a calcium salt or a manganese salt may be used. The metal salt to be used is preferably soluble in water at pH 3.5 or below.
The metal salt can take various forms such as a chloride, a sulfate or a nitride, when used. The concentration of the inorganic salt to be added is from 0.1 to 10 wt %, preferably from 0.2 to 2.0 wt %.
It is not favorable for preparation of a HA gel with fluidity that the concentration is below 0.1 wt %, because the resulting gel tends to be solid. It is not favorable that the concentration is over 10 wt %, because gelation takes an impractically long time.
The pH of a HA aqueous solution is adjusted so that a sufficient proportion of the carboxylic groups in HA undergoes protonation. In the present invention, it is necessary to adjust the pH to 3.5 or below, preferably to 2.5 or below, although the final pH is set depending on various factors such as the type of the counterinon in the HA salt, the molecular weight of HA, the concentration of the aqueous solution, conditions of freezing and thawing and the properties of the resulting gel such as strength.
With respect to freezing-thawing, a procedure comprising freezing the prepared acidic HA aqueous solution in an appropriate vessel at a predetermined temperature and then thawing it at a predetermined temperature is carried out at least once.
Although the freezing and thawing temperatures and times may be appropriately set depending on various factors such as the size of the vessel, the volume of the aqueous solution, the molecular weight of HA, the concentration of the aqueous solution, the pH of the aqueous solution and the concentration and kind of the metal salt in it so that the acidic HA solution freezes and thaws, it is generally preferred that the freezing temperature is not higher than the ice point, and the thawing temperature is not lower than the ice point.
It is particularly preferred that the freezing temperature is xe2x88x925xc2x0 C. or below, and the thawing temperature is 5xc2x0 C. or above, to shorten the freezing and thawing times. There is no restriction on the freezing and thawing times so long as they are longer than it takes to complete freezing and thawing at the temperatures.
The number of times the procedure comprising freezing and then thawing the prepared acidic HA aqueous solution is repeated, depends on various factors such as the molecular weight of HA to be used, the concentration of the metal salt in it, the concentration and pH of the aqueous solution, the freezing and thawing temperatures and times and the properties of the resulting gel such as strength. Usually, it is preferred to repeat the procedure at least once.
Further, the freezing and thawing temperatures and times may be changed every time the freezing-thawing is repeated.
From the HA gel obtained by freezing and thawing a prepared acidic HA aqueous solution, the acid component added for the acidification has to be removed in order to prevent acid hydrolysis of HA. For removal of the acid component, the gel is usually washed with or dialyzed against an aqueous solvent, for example, water, physiological saline or a phosphate buffer, preferably physiological saline or a phosphate buffer. There is no restriction on the aqueous solvent so long as it does not functionally impair the HA gel.
Although there is no particular restriction on the method for washing or dialysis, dialysis is preferable. Dialysis is accomplished preferably by using a dialysis membrane or a ultrafilter. The dialysis conditions, inclusive of the volume of the solvent and the number of times of dialysis, are determined so that the concentration of the component to remove can be lowered to the desired level or below. The pH pf the dialyzed gel is adjusted to meet the purpose before use.
The HA gel of the present invention is obtainable endotoxin-free and aseptically if care is taken over the reagents, water, the vessels.
The HA gel thus prepared has fluidity by itself and is obtained with uniformity and transparency but without turbidity. It may be filled into a syringe or a bag before use. If pharmaceutically or physiologically active substances are added at the time of gelation, the resulting fluid HA gel contains these substances in it.
For example, addition of thrombin which coagulates blood by catalyzing conversion of fibrinogen into fibrin in the blood coagulation cascade with a view to accelerating embolization and addition of various antitumor agents with a view to obstructing tumor arteries may be mentioned without any restriction.
The HA gel of the present invention shows great improvement in in vivo residency and persistency over HA solution and excellently safe and biocompatible by virtue of the absence of crosslinkers. Therefore, it can be used as a biomedical material such as an injection for treatment of arthrosis, an embolizing material, an injection for a soft tissue and an artificial vitreous body.